The present invention concerns a biochemical sensor system whose sensitivity is increased by molecular amplification of a signal initialised by the interaction between a biochemical entity present in a solution or a biological fluid and a reagent immobilised on the substrate of the sensor and having a specific affinity for said biochemical entity.
In the field of biological sensors, systems are increasingly sought which allow the limits of detection and analysis of biological entities in biotic fluids to be pushed back further, in the hope of obtaining very high detection sensitivity. Thus, technological improvements have concerned not only the instrumental environment, for example the limits for detecting a signal, but also the design of the sensor itself as soon as limits of sophistication were attained at the instrumental level. But even here, improvements at the sensor level have reached a threshold beyond which it is no longer possible to detect the biomolecules in the form of traces such threshold being of the order of a nanomolar (nM) or picolmolar (pM).
Nonetheless, other improvements have enabled the sensor signal, until then undetectable by former technologies, to be measured owing to an amplification of the signal on which the detection principle relies. Such amplification finds its preferred application in the field of biological sensors given that the conditions implemented in biological analysis are compatible with bio-amplification systems.
Currently, two bio-amplification modes are employed in systems intended to detect, for example, immunological reactions. According to a first amplification mode, in ELISA (Enzyme Linked Immunosorbent Assays) tests, the molecule to be detected, for example an antibody which interacts with a chemical entity such as an immobilised antigen, is chemically linked to an enzyme. The enzyme is used to catalyse the transformation of the detectable molecules. In the currently used ELISA systems, the enzymes which catalyse the production of chemical entities are almost always hydrolases. The water soluble reaction products are preferably detected in the whole reactional medium by measuring absorption, luminescence or bioluminescence.
In biosensors, a second amplification mode is obtained by increasing the number or mass of species detected. This amplification principle is achieved for example by linking mass markers to the molecule to be detected.
If the detection principle relies on fluorescence or absorption, fluorescent or absorbent molecules are chemically linked to the chemical entity. By way of an example of this type of amplification, U.S. Pat. No. 5,175,270 may be cited, which discloses an amplification mechanism from a dendrimer architecture at the surface of the sensor. The modified molecule link on each molecule targeted, or the marked secondary reagent link (for example colloids, nanoparticles or fluorophore-labelled secondary antibodies will produce linear signal amplification. Latex balls, semiconductor nanocrystalline compounds or colloidal gold are mass markers currently used in biosensor systems. In commercial amplification systems, secondary antibodies strongly marked by fluorescent molecules contribute to increasing the signal in a linear manner.
Generally, the usual systems amplify the sensor signals via reactions catalysed by an enzyme which increases the number of secondary chemical entities in the medium by catalysis (catalytic amplification for global detection). Otherwise, the sensor signals are increased either by adding mass, for mass sensitive detection, or by increasing the number of labelled molecules which are linked to the unit.
By way of example of linear amplification at the surface of a sensor, fluorescent signal amplification may be cited: in this system the secondary antibodies are conjugated to allow detection of targets in small quantities.
These two amplification modes which have just been briefly described have allowed detection sensitivity to be substantially increased, either in solution or on a surface: they do not however allow a sufficiently high signal to be obtained to make them able to be used sufficiently in practice.